Foodborne pathogenic bacteria in wild European hedgehogs (Erinaceus europaeus)

Background European hedgehogs (Erinaceus europaeus) are widely distributed across Europe. They may play an important role by spreading zoonotic bacteria in the environment and to humans and animals. The aim of our work was to study the prevalence and characteristics of the most important foodborne bacterial pathogens in wild hedgehogs. Results Faecal samples from 148 hospitalised wild hedgehogs originating from the Helsinki region in southern Finland were studied. Foodborne pathogens were detected in 60% of the hedgehogs by PCR. Listeria (26%) and STEC (26%) were the most common foodborne pathogens. Salmonella, Yersinia, and Campylobacter were detected in 18%, 16%, and 7% of hedgehogs, respectively. Salmonella and Yersinia were highly susceptible to the tested antimicrobials. Salmonella Enteritidis and Listeria monocytogenes 2a were the most common types found in hedgehogs. All S. Enteritidis belonged to one sequence type (ST11), forming four clusters of closely related isolates. L. monocytogenes was genetically more diverse than Salmonella, belonging to 11 STs. C. jejuni ST45 and ST677, Y. pseudotuberculosis O:1 of ST9 and ST42, and Y. enterocolitica O:9 of ST139 were also found. Conclusions Our study shows that wild European hedgehogs should be considered an important source of foodborne pathogens, and appropriate hygiene measures after any contact with hedgehogs and strict biosecurity around farms are therefore important.

introduced by humans to new areas due to the assumption that the species kills snakes and rats [2].European wild hedgehogs are protected by legislation in most European countries, including Finland, and keeping them as pets is therefore illegal [3].They are nocturnal hibernators that are active during both night and day in cities. Hedgehogs are permanent residents of parks and gardens, seeking food and shelter.Householders may provide them with various refugia and supplementary food in their gardens.The European hedgehog is an insectivore that readily eats food offered by humans.Most wild hedgehogs in Finland are killed by traffic or die by starvation [4].

Background
The European hedgehog (Erinaceus europaeus) is a common hedgehog species, which is widely distributed across Europe.In Finland, they live at the northernmost limit of their distribution range [1].Hedgehogs in Finland, which apparently originate from Estonia and Russia, have been Zoonotic bacteria play an important role in the interactions between humans, domestic animals, and wildlife [5].Wild hedgehogs in (sub)urban areas have been shown to carry a number of zoonotic agents and may contribute to their spread [6,7].Thermotolerant Campylobacter spp., Salmonella enterica, enteropathogenic Yersinia spp.(Y.enterocolitica and Y. pseudotuberculosis), shigatoxinproducing Escherichia coli (STEC), and Listeria monocytogenes are the most common zoonotic bacteria causing foodborne infections among humans in Europe [8].These bacteria have sporadically been found in wild hedgehogs in Europe [3,5,7,9,10].Salmonella spp. in particular has been found in both wild and pet hedgehogs [4,11,12].Salmonella infection in hedgehogs may be a latent infection but Salmonella can also cause enteritis, even sudden death, especially in young animals [12].Hedgehogs have also been linked to human salmonellosis outbreaks [11,13,14].However, human infections due to wildlife contact have rarely been reported [15].
Information on the presence and characteristics of foodborne pathogenic bacteria in hedgehogs is scarce.In this study, we screened the prevalence of the most important foodborne bacteria in the faeces of (sub)urban European hedgehogs using PCR and bacterial culture.We also characterized the isolates using several methods, including whole-genome sequencing, to gain more information on the characteristics of these pathogens.

Samples and sample preparation
In total, 148 wild hedgehogs were studied between 2020 and 2022.The hedgehogs originated from the Helsinki region, southern Finland.They were brought to the wildlife hospital of Korkeasaari Zoo due to injuries or diseases, where they were either euthanized or died due to the severity of their conditions.Frozen carcasses of hedgehogs were transported from the zoo to the laboratory.Hedgehogs were categorized as young or adult, based on their size and body weight.Individuals with small size and body weight below 500 g were considered young.

PCR detection of pathogenic bacteria
Real-time PCR was used to study the presence of genes carried by Campylobacter (rrn) [16], Salmonella (ttr) [17], enteropathogenic Yersinia (ail) [18,19], STEC (stx1 and stx2) [20], and Listeria (mpl) [21].A rectal sample of faecal matter from each hedgehog was taken with a cotton swab and mixed well with 10 ml of buffered peptone water (BPW, Oxoid, Basingstoke, UK).DNA was extracted from 1 ml of overnight enriched BPW using a Quick-DNA Fecal/Soil Microbe Miniprep kit (Nordic BioSite Oy, Helsinki, Finland).Two µl of the DNA was added to 18 µl of PCR mix consisting of 1x ready-to-use mix (iQ SYBRGreen Supermix or SsoAdvanced Universal SYBRGreen Supermix, Bio-Rad) and 200 nM of primers (metabion, Planegg, Germany).A three-step protocol (denaturation at 95 °C for 10 s, annealing at 58 °C for 10 s, and elongation at 72 °C for 30 s with 40 cycles followed by a melting curve analysis was used.The fluorescence intensity of SYBR Green was studied using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with CFX Maestro Software v.2.3.A sample was considered positive when the threshold cycle (Ct) was below 38 and a specific melting temperature (Tm) was observed.

Culturing of pathogenic bacteria
One hundred µl of overnight enrichment of PCR-positive Salmonella, Yersinia, and Listeria samples were plated on selective CHROMagar Salmonella Plus (37 °C, 24 h) (Labema, Kerava, Finland), Yersinia C.I.N Agar (30 °C, 24 h) (Oxoid), and Listeria Chromogenic Agar ALOA (37 °C, 24 h) (Labema) agar plates, respectively.Campylobacter presence was studied from all samples by direct plating on selective mCCDA plates (microaerophilic at 41.5 °C, 48 h) (Oxoid).Typical colonies on all selective agar plates were identified by real-time PCR using the same primers as in the PCR screening, with a threshold cycle below 30.

Whole-genome sequencing
DNA of Salmonella, Campylobacter, Yersinia, and Listeria isolates was extracted using PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbaden, CA, USA).DNA quality was measured by NanoDrop (ThermoFischer Scientific, Waltham, MA, USA), based on the 260/280 ratio, and DNA quantity by Qubit (ThermoFischer Scientific).Whole-genome sequencing (WGS) was performed for Salmonella, Yersinia, and Listeria on the Illumina DNA platform by CeGaT (Center for Genomics and Transcriptomics, Tuebingen, Germany).An Illumina DNA Prep library preparation kit and NovaSeq6000 were used to generate 100 bp paired-end reads.The short reads were assembled de novo using a Unicycler v.0.4.8 assembler available on the PATRIC 3.6.12platform (https://www.bv-brc.org/app/Assembly).WGS of Campylobacter was performed at the Institute for Molecular Medicine Finland or Novogene (Cambridge, UK) using NoveSe-qPE150.Raw sequence data were assembled using the INNUca pipeline (https://github.com/B-UMMI/INNUca).

Statistical analyses
The analyses were performed with IBM SPSS Statistics 29.0.1 (IBM, Armonk, NY, USA).All tested variables were categorical.Fisher's exact test was used to compare the differences between the proportions of two groups.
Foodborne pathogens were frequently detected in the faeces of 148 hedgehogs from the Helsinki region (Table 2).In total, 89 (60.1%) of the hedgehogs were positive for at least one foodborne pathogen species in their faeces.Listeria (26.4%) and STEC (25.7%) were the most common pathogens detected in the faeces by PCR.Salmonella and Campylobacter were detected in 17.6% and 6.8% of the hedgehogs, respectively.Enteropathogenic (ail-positive) Yersinia was detected in 16.2% of the  The prevalence of foodborne pathogens in the faecal samples varied between young animals and adults (Table 3).Salmonella was detected more often (P = 0.05) in young animals (25.0%) than in adults (11.9%), while STEC was more commonly (P = 0.02) found in adults (33.3%) than in young animals (15.6%).In 2022, ail-positive Yersinia was detected significantly (P = 0.04) more often in young hedgehogs (61.5%) than in adults (23.8%).No significant differences were observed between the pathogen prevalences in injured and sick hedgehogs.The prevalences of most pathogens differed clearly between the sampling years (Table 3).Only Listeria was quite evenly distributed during the years; its prevalence varied between 25.0% and 29.4%.Salmonella was most (26.8%)frequently detected in 2021, while Yersinia (38.2%) and STEC (47.1%) were frequent findings in 2022.Significant differences were observed between the sampling years in the prevalence of Yersinia and STEC (Table 3).
In total, 29 S. Enteritidis and 2 S. Typhimurium isolates were characterized with Ridom cgMLST.Most (99.7%, 26/29) S. Enteritidis isolates formed four clusters, with 0 to 5 ADs in each cluster (Fig. 1).Most isolates (11/14) in the largest cluster were highly related, with no ADs, including 14 S. Enteritidis isolates (S1-S14).These isolates were found from hedgehogs sampled during different years and from different areas in the Helsinki region (Table 5).The two S. Typhimurium isolates differed from each other with 426 allelic differences.
In total, 29 L. monocytogenes isolates were analysed with Ridom MLST.Eleven (37.9%)L. monocytogenes 1/2a isolates formed two clusters with 1 to 9 ADs (Fig. 2).The largest cluster, with 1 to 8 ADs, was formed by L. monocytogenes isolates (L21-L29) belonging to the sequence type ST2488.These isolates originated from hedgehogs sampled during different years and from different areas (Table 5).Only two isolates (L7 and L8) with 9 ADs belonged to ST398 in the second cluster.These two isolates originated from hedgehogs sampled in 2020  indicating that the two strains were clearly distinct and not closely related to each other.

Discussion
Our study shows that wild European hedgehogs are an important source of foodborne zoonotic bacteria.We detected the zoonotic bacteria by PCR, which is a very sensitive method, detecting small numbers of bacteria [26].Approximately 60% of the hedgehogs excreted at least one pathogenic bacterial species in their faeces.The faeces samples were collected from carcasses of sick or injured hedgehogs, which may have affected the high prevalence.The most frequently detected species were L. monocytogenes and STEC.Both pathogens have rarely been reported in hedgehogs before; however, only a few studies have been conducted previously [5,9].We isolated L. monocytogenes from 20% of the faecal samples.
In an earlier study, this pathogen was found in extraintestinal sites from 2% of the tested hedgehogs [9].We did not find any significant temporal or regional differences in Listeria prevalence.We also found no significant difference between young and adult animals.Our study demonstrates that L. monocytogenes is commonly excreted in the faeces of hedgehogs.L. monocytogenes is ubiquitous in the environment [27], which indicates that hedgehogs can contract this pathogen from the environment through eating soil-dwelling invertebrates.The high PCR prevalence of STEC in the hedgehogs was interesting; however, this topic requires further study, as we did not have any isolates for characterization, which is needed for estimating isolate pathogenicity.STEC has not been reported in hedgehogs before.Salmonella was detected in the faecal samples of 26 (18%) hedgehogs by PCR, and it was also isolated from all these samples, indicating that the number of Salmonella in the faeces was quite high.A prevalence of 18% is high in countries like Finland, where Salmonella prevalence is very low (< 1%) in meat-producing animals (pigs, ruminants, and poultry) [28].One reason for the high prevalence in the wild hedgehogs may be that our samples originated from injured and sick animals.However, the high prevalence was not a surprise because Salmonella has been isolated from 57% of dead hedgehogs in Finland in an earlier study [4].Reasons behind the very high prevalence in this earlier study are probably due to the very restricted sampling area and because liver samples were used.Salmonellosis in hedgehogs is assumed to mostly be a latent infection found in the liver [12].Salmonella prevalence in the faeces of European hedgehogs in Europe has been low (3-4%) in earlier studies [5,29].
One reason for the high Salmonella prevalence in the hedgehog faeces in our study may be that the animals originated from (sub)urban areas.Urban hedgehogs have been suggested to carry more zoonotic pathogens than those in rural areas [30].All the hedgehogs in our study had been injured or sick, which is a stress factor that may have activated the latent infection and increased the excretion of Salmonella in their faeces.Salmonella was also significantly more often detected in young hedgehogs than in adults.
Our study showed that wild European hedgehogs are also possible carriers of Campylobacter and pathogenic (ail-positive) Yersinia.However, both pathogens were quite rarely isolated from the faecal samples.Only a few earlier studies [7,10,31] have reported C. jejuni and Y. pseudotuberculosis in hedgehogs.Using PCR, we detected Y. pseudotuberculosis (14%) more frequently than Y. enterocolitica (3%) in the faecal samples.Wild animals are assumed to be the most important reservoir for Y. pseudotuberculosis [32].Isolation of Yersinia, especially, Y. pseudotuberculosis is very challenging, which may be one reason for the low isolation rate.
We only found two Salmonella serotypes in hedgehog faeces: Enteritidis and Typhimurium.These serotypes are the most prevalent serotypes responsible for human salmonellosis in the EU [8], and they are also the most frequent serotypes reported in hedgehogs [3,7,13,29].In Denmark, S. Enteritidis isolates found in hedgehogs were shown to belong to the same clonal lineage as isolates found in infected humans [33].Enteritidis was frequently found in wild hedgehogs in our study.It was the only serotype found in the hedgehogs in Finland in a previous study [4].All Enteritidis isolates found in our study belonged to ST11.This type was reported to be common in hedgehogs and humans in the UK [29].In Germany, S. Enteritidis ST183 was associated with hedgehogs [15].  5. Number of allelic differences (ADs) between the isolates are indicated on the connecting lines.Clusters are shaded in grey and a cluster distance threshold of maximum 10 ADs was used Most (90%) of our S. Enteritidis ST11 isolates formed four clusters with closely related isolates.Interestingly, most (79%) isolates in the largest cluster were highly related, with no ADs, even if they differed temporally and geographically from each other.This indicates that we have endemic S. Enteritidis isolates circulating among hedgehogs in the Helsinki region, despite the hedgehogs not necessarily being epidemiologically linked to each other.In two hedgehogs, we also identified Typhimurium, which belonged to ST19 and clearly differed (426 ADs) from each other.Hedgehogs are suggested to be a reservoir for S. Typhimurium, and they have also been linked to two human salmonellosis outbreaks caused by Typhimurium in Norway [13].Outbreaks due to S. Typhimurium have also been linked to pet hedgehogs [11,14,34].We could not show any antimicrobial resistance among Salmonella isolates in our study.Also, no evidence of antimicrobial resistance was present in the hedgehog Enteritidis isolates in the UK [29].
L. monocytogenes isolates recovered in our study belonged to serogroups 2a and 4b, which are commonly found in animals and humans.These types have also been identified in wild animals in Finland [35] and from the livers of wild hedgehogs in the UK [9].We found several STs among serotype 2a and 4b isolates, especially among 2a isolates, which dominated in the hedgehogs in our study.
Most of the STs identified in this study have been found in wild animals in our earlier study, indicating that these STs (ST1, ST4, ST7, ST18, ST37, ST451, and ST585) circulate in wildlife in southern Finland [35].Interestingly, several isolates belonging to ST2488 formed a cluster with only 1 to 8 ADs.These isolates mostly differed from each other both temporally and regionally.This sequence type could be an endemic type adapted to hedgehogs in the Helsinki region.
We identified three C. jejuni isolates, which belonged to ST45 and ST677.Both types have been predominant among domestically acquired human infections [36,37] and in broilers in Finland [38].In addition, ST45 has been found from various other sources, including cattle, sheep, pigs, cats and dogs, wild birds, and environmental waters worldwide (PubMLST Campylobacter jejuni/coli isolate database) [39].On the other hand, ST677 has been linked to more severe bacteraemia cases, in addition to gastroenteritis, in Finland [40].Although mainly found in humans, ST677 has also been found, albeit infrequently, from other sources, with wild birds and chickens likely being the main reservoirs.We also found C. lari ST21 from one hedgehog.Overall C. lari is an uncommon finding in human infections compared to C. jejuni and C. coli [8].However, ST21 has been identified as the most prevalent genotype among human C. lari infections in Europe [41] and the USA [42].It has also been found from various sources (animals, food, and environment among others) [41].Overall, wild birds, especially seagulls, and shellfish are considered to be the main reservoirs of C. lari [43].Although clinically relevant Campylobacter spp.and genotypes were identified among hedgehogs in our study, they were relatively infrequent findings.Furthermore, only a few samples were culture positive, suggesting that hedgehogs are unlikely to play a major role in the epidemiology of Campylobacter species in humans.Y. pseudotuberculosis isolates found in three hedgehogs were identified as ST9 (O:1b) and ST42 (O:1a), and they were not closely related to each other based on cgMLST.However, both STs are common types in wildlife and have also been found in wild and domestic animals in Finland [44,45].Additionally, one pathogenic (ail-positive) Y. enterocolitica was found and it was identified as O:9, which is the second most common serotype found in humans [8].This serotype is associated with ruminants and has been found in sheep and voles in Finland [46,47].The ail-positive Yersinia isolates were susceptible to almost all the antimicrobial agents tested in our study.The Y. enterocolitica O:9 isolate was only resistant to ampicillin.Y. enterocolitica is naturally resistant to this agent due to β-lactamase production.Y. pseudotuberculosis O:1 isolates were resistant only to colistin.Colistin resistance has been shown to be common in Y. pseudotuberculosis due to mutations in genes responsible for their lipopolysaccharide structure [48].
Wild European hedgehogs, especially stressed or sick ones, may contribute to the spread and transmission of important foodborne pathogens due to the high prevalence in their faeces.As hedgehogs often live in (sub) urban areas, people handling and feeding hedgehogs may expose themselves to foodborne pathogens carried by hedgehogs.A high prevalence of Salmonella, especially among hedgehogs sampled at feeding places, has been reported in Norway [13].The same study suggested that hedgehog feeding places may serve as sites of Salmonella transmission between animals.Appropriate hygienic measures after any contact with hedgehogs are recommended to reduce the risk of enteric pathogen transmission from hedgehogs to humans [34].Hedgehogs inhabiting parks and gardens with close contact to humans and other animals may pose a public health hazard that needs to be investigated further.

Conclusions
Our results suggest that wild European hedgehogs should be considered an important source of foodborne zoonotic bacteria.S. Enteritidis ST11 was isolated from the faeces of several injured and sick wild hedgehogs originating from various parts in the Helsinki region, indicating that this pathogen is endemic and widely distributed in European hedgehogs in southern Finland.Salmonella monitoring in hedgehogs around farms, especially in countries with low Salmonella prevalence in farm animals, could be one way of obtaining information on the Salmonella situation around farms.Strict farm biosecurity is crucial to prevent Salmonella and other foodborne pathogens from entering the food production chain.

Fig. 1
Fig. 1 Minimum spanning tree of 31 Salmonella isolates from wild hedgehogs in southern Finland during 2020-22.Nodes are numbered according to Table 5. Number of allelic differences (ADs) between the isolates are indicated on the connecting lines.Clusters are shaded in grey and a cluster distance threshold of maximum 10 ADs was used

Fig. 2
Fig. 2 Minimum spanning tree of 29 Listeria monocytogenes isolates from wild hedgehogs in southern Finland during 2020-22.Nodes are numbered according to Table 5. Number of allelic differences (ADs) between the isolates are indicated on the connecting lines.Clusters are shaded in grey and a cluster distance threshold of maximum 10 ADs was used

Table 1
Number of hedgehogs from the Helsinki region (southern Finland) studied between 2020 and 2022 a A maximum radius of 65 km from Helsinki City

Table 2
Detection of foodborne pathogens in 148 wild hedgehogs in southern Finland by PCR

Table 3
Distribution of foodborne pathogens among 148 wild hedgehogs depending on the grouping Superscripts a and b differed significantly (P ≤ 0.05) from each other

Table 4
Multi-locus sequence types (MLSTs) identified among foodborne pathogenic bacteria isolated from hedgehogs between 2020 and 2022 in Helsinki region, southern Finland a Serogroup

Table 5
Salmonella enterica and Listeria monocytogenes isolates characterized with cgMLST a Sequence type based on seven housekeeping genes b Allelic differences c A maximum radius of 65 km from Helsinki City d From the same animal e Serogroup